A highly active soluble diacylglycerol synthesizing system from developing rapeseed, Brassica napus L.

The subcellular distribution of the enzymes of triacylglycerol biosynthesis has been studied in developing oilseed rape. All in vitro enzymatic activities from oleoyl-CoA to triacylglycerol were sufficient to account for the known rate of oleate deposition in triacylglycerol in vivo. The enzymatic activities from oleoyl-CoA to diacylglycerol preferentially were localized in a 150,000 g supernatant fraction, while the diacylglycerol acyl-transferase mostly was associated with the microsomal (20,000 g pellet and 150,000 g pellet) and oil-body fractions. The soluble (150,000 g supernatant) fraction rapidly incorporated oleate from [1-14C]oleoyl-CoA into diacylglycerol with rates of 40 nm min-1 g-1 FW at 20 microM oleoyl-CoA. The pH optimum was 7.5-9.0, and normal saturation kinetics were seen with oleoyl-CoA; the S0.5 was about 32 microM. Exogenous acyl acceptors, such as glycerol 3-phosphate, lysophosphatidic acid and lysophosphatidyl-choline stimulated oleate incorporation into diacylglycerol. The detergents Triton X-100 and sodium cholate inhibited diacylglycerol formation at concentrations in the region of their critical micellar concentration, while n-octyl-beta, D-glyco-pyranoside had no effect, even at high concentration. The significance of these findings for the mechanism of oil-body formation in developing oilseeds is discussed.